The adhesion and migration of adherent cells depends on the function of cell-surface glycoproteins that mediate the adhesive contact between the cell surface and molecules in the cellular microenvironment. In quiescent, non-migratory cells, adhesion receptors are responsible for maintaining stable contact between the cell and its environment. As cells translocate, adhesion proteins transduce traction forces from the cytoskeleton across the cytoplasmic membrane to the substrate, permitting cells to pull against their environment. Thus, receptor-mediated traction-force generation depends on connections between adhesion receptors and the cytoskeleton. In addition, receptor-cytoskeleton interactions play a crucial role in the stabilization of adhesive complexes thus modulating binding avidity.
The establishment and maintenance of neuronal connections are essential features of nervous system function. The activity of adhesion proteins on the cell surface is essential to both of these processes. During development, the translocation of the neuronal growth cone depends critically on adhesion proteins that mediate the recognition of molecules in the extracellular environment (Tessier-Lavigne and Goodman. Science 1986;274:1123-1133). In the adult, many of the same adhesion proteins help maintain axon fascicles and synaptic contacts. Whereas receptors involved in adhesive contacts mediate largely static connections between adjacent cells, adhesion receptors in migrating cells serve to elaborate cellular traction forces between the cell and its environment (Harris et al. Science 1980;208: 177-179 and Galbraith and Sheetz J. Cell Biol 1999; 147:1313-1324). A variety of adhesion receptor families have been shown to serve as receptors for permissive, substrate-bound molecules that promote axon outgrowth, including integrins, Ig-CAM's and cadherins (Kamiguchi and Yoshihara J. Neurosci 2001;21:91941-9203). This common activity raises the possibility of a common traction-based mechanism for transducing permissive cues. However, while the biophysical basis of integrin-mediated traction-force generation and adhesion is fairly well characterized (Felsenfeld et al. Nature 1996;383:438-440; Choquet et al. Cell 1997;88:39-48; Galbraith and Sheetz. Proc Natl Acad Sci USA 1997;94:9114-9118; and Yauch et al. J Exp Med 1997;186:1347-1355), little is known about the differential regulation of adhesion and migration in other families of adhesion receptors.
Immunoglobulin-family cell adhesion molecules (Ig-CAMs) have been implicated in the guided growth of neuronal processes during development (Kamiguchi and Lemmon. Curr Opin Cell Biol 2000; 12:598-605 and Rutishauser J Cell Biol 2000; 149:757-760). In the vertebrate central nervous system, L1-CAM, an Ig-CAM, plays an essential role in the growth and guidance of axons towards their targets (Daline et al. Nat Genet 1997;17:346-349 and Cohen et al. Curr Biol 1998;8:26-33). L1-CAM mutations in humans lead to a variety of developmental defects including corpus callosum hyperplasia, mental retardation, adducted thumbs, spastic paraplegia and hydrocephalus (CRASH syndrome), suggesting that L1-CAM plays a crucial role in the development of the central nervous system (Fransen et al. Eur J Hum Genet 1995;3:273-284). Moreover, the capacity of substrate-immobilized L1-CAM-ligands to promote neurite extension in vitro through interactions with cell-surface L1-CAM (Lemmon et al. Neuron 1989;2:1597-1603; Kuhn et al. J Cell Biol 1991;115:1113-1126; and Felsenfeld et al. Neuron 1994; 12:675-690) raises the possibility that L1-CAM on the growth cone may mediate the generation of traction forces in a mechanism similar to that observed for integrins in other cell types. In addition, L1-CAM may play other roles in the development and maintenance of the nervous system, including the stabilization of axon fascicles in the mature animal (Dahme et al. Nat Genet 1997;17:346-349). To accomplish each of these diverse functions, the movement of L1-CAM in the plane of the membrane must be regulated across the cell surface.
The regulation of adhesion receptor distribution, movement, and function in adhesion and migration depends on the connection between these glycoproteins and components of the cytoskeleton. L1-CAM interacts with as many as four cytosolic binding partners through two discrete sites in the cytoplasmic tail (Kamiguchi et al. J Neurosci 1998; 18:5311-5321; Zhang et al. J Biol Chem 1998;273:30785-30794; Dickson et al. J Cell Biol 2002;157:1105-1112; and Kizhatil et al. J Neurosci 2002;22:7945-7958). The binding of L1-CAMs to members of the ankyrin family of cytoskeletal adaptor proteins is perhaps the best characterized of these interactions (Davis and Bennett J Biol Chem 1994;269:27163-27166; Garver et al. J Cell Biol 1997;137:703-714; and Hortsch et al. Cell Adhes Commun 1998;5:61-73). In vertebrates, three distinct genes encoding ankyrin family members, ankyrinB, ankyrinG and ankyrinR, have been identified with distinct but overlapping expression patterns (Bennett and Baines Physiol Rev 2001;81:1353-1392). In the nervous system and heart, ankyrins appear to play a crucial role in the organization of cellular structures involved in signaling (Lambert et al. J Neurosci 1997; 17:7025-7036 and Tuvia et al. J Cell Biol 1999; 147:995-1008). The L1-CAM family member neurofascin binds to ankyrin through a motif that is highly conserved among L1-CAM-family members near the carboxy-terminus of the cytoplasmic tail (Garver et al. J Cell Biol 1997; 137:703-714). The ankyrin binding site, mapped based on the interaction between neurofascin and ankyrinG, is comprised of a 12 amino acid motif, QFNEDGSFIGQY (SEQ ID NO: 1), which includes a carboxy-terminal tyrosine and is identical in neurofascin and L1-CAM from rat (Miura et al. FEBS Lett 1991;289:91-95 and Zhang et al. J Biol Chem 1998;273:30785-20794). Ankyrin binds to this motif in its dephosphorylated state (Garver et al., supra). In addition, when phosphorylated, this site serves as a binding partner for the protein doublecortin (Kizhatil et al., supra), a protein which has been implicated in the migration of neuronal progenitors to their proper lamina in the mature cerebral cortex (Francis et al. Neuron 1999;23:247-256 and Gleeson et al. Neuron 1999;23:257-271). Mutations at this site in human L1-CAM lead to a similar disruption in ankyrin binding (Needham et al. J Neurosci 2001;21:1490-1500). However, the drosophila L1-CAM homolog neuroglian, while requiring the FIGQY (SEQ ID NO: 8) motif for ankyrin recruitment, appears to be regulated primarily through ligation of the extracellular domain (Hortsch et al., supra). At a functional level, the binding of ankyrin to L1-CAMs like neurofascin plays a critical role in L1-CAM-mediated cell adhesion (Tuvia et al. Proc Natl Acad Sci USA 1997;94:12957-12962).
In addition to ankyrin binding, a distinct phosphorylation site (YRSLE; SEQ ID NO: 7) upstream of the ankyrin site binds both the μ2 chain of the AP-2 clathrin complex (Kamiguchi and Lemmon J Neurosci 1998;18:3749-3756 and Schaefer et al. J Cell Biol 2002; 157: 1223-1232) and ERM proteins (Dickson et al. J Cell Biol 2002; 157:1105-1112). The binding to AP-2 appears to play a critical role in the endocytosis and recycling of L1-CAM at the back of the growth cone, a process essential to the function of L1-CAM in growth cone migration (Kamiguchi and Yoshihara. J Neurosci 2001;21:9194-9203).
Axonal damage characterizes diseases such as spinal cord injury, traumatic brain injury, stroke, and neurodegenerative disease. Spinal cord injury affects millions of individuals worldwide, resulting in severe impairment of the physical function of affected persons (e.g., as seen in paraplegia and quadriplegia). Traumatic brain injury is a major health problem in all developed countries. Stroke is the second largest cause of death worldwide, and the main cause of long-term neurological disability. Neurodegenerative disease is of increasing concern with the aging of the population of the developed world. Currently available therapies are unable to repair the axonal damage. Therefore, the need exists in the art for therapies to repair neuronal damage, which therapies depend upon the ability to promote the outgrowth of spinal cord neurons to reestablish the damaged neuronal connections.
The outline of the sensory pathway is well known in the art. From the source of pain, pain messages move through peripheral sensory neurons and up the dorsal root ganglion of the spinal cord, where they stimulate interneurons in a relay destined for the brain. Pain messages can be blocked, enhanced, or modified at the relay between the peripheral neuron and the interneuron before progressing to the brain. From the spinal cord, the signal reaches the thalamus and cortex of the brain, where the location, intensity, and nature of the pain is decoded. Once the brain has interpreted the pain signal, the brain sends pain-suppressing chemicals to the pain source and triggers other related responses.
Neurotransmission between cells can be regulated by voltage-gated calcium channels, which mediate calcium influx in response to membrane depolarization. Voltage-dependent calcium channels are the primary trigger for electrically stimulated release of chemical transmitters in the nervous system that lead to stimulation of specific neuronal pathways. Electrical currents are used in neurons to rapidly transmit signals. All cells have a resting potential: an electrical charge across the plasma membrane, where the exterior of the cell is positive and the interior negative, due to the concentrations of different populations of charged ions. In neurons, voltage gated channels open or close in an “all-or-none” fashion in response to changes in the charge (measured in volts) across the plasma membrane of the cell. Activity- and receptor-dependent redistribution of ionotropic receptors has been widely studied in the post-synaptic density (Zhu J J et al., Cell. 2002 Aug. 23; 110(4): 443-55. Rao A, Craig A M., Neuron. 1997 Oct.; 19(4): 801-12.), but such studies have not previously been extended to proteins in the presynaptic active zones.
Dunlap and Fischbach (Dunlap K, et al. J. Physiol. 1981, 317: 519-5335) have suggested that transmitter-mediated shortening of the duration of the action potential could be due to a decrease in calcium conductance or a decrease in the number of functional channels in the membrane. Inhibition of Ca2+ channels can be voltage-dependent, and is mediated by G protein beta-gamma subunits (Ikeda S R. Nature. 1996 Mar. 21; 380(6571): 255-8.; Herlitze S, et al. Nature. 1996 Mar. 21; 380(6571): 258-62.). In addition, kinases such as protein kinase C and tyrosine kinases have been shown to inhibit Ca2+ channels (Hille B. Trends Neurosci. 1994 Dec.; 17(12): 531-6). Subsequent work has established that G protein-dependent inhibition of calcium current is in part a result of a decrease in the open probability of the channel, reducing current density (Delcour, Ah, and Tsien R W. 1993. Science February. 12, 259 (5097):989-4.). The idea that changes in channel density could underlie calcium channel modulation has not previously been tested. Alteration of a presynaptic calcium channel plasma membrane density to modulate calcium channel function will be useful in the control of neuronal signal propagation.
Chronic pain affects millions of individuals; it results in severe impairment of a patient's physical function and is often associated with psychological depression. Currently available therapies teach the use of analgesics to treat the symptoms of pain that result from the activation of pain pathways. These analgesics become less effective over time as patients become sensitized. Therefore, the need exists in the art for novel therapies for chronic pain that block pain pathway signals before they reach the brain.
Stroke and traumatic head injury result in similar pathological processes in the brain. In traumatic head injury, neurons die because they are crushed, setting off an inflammatory cascade that triggers further neuronal apoptosis. In stroke, the infarct results a similar cascade in the brain. Progressive inflammation and cell death downstream of the primary event is mediated by calcium flux across the neuronal cell membrane. Calcium flux through L type channels across the neuronal membrane and release from calcium stores leads to apoptosis. By blocking the calcium flux, one may block the cell death resulting from a or stroke traumatic head injury (Mattson M P, et al., Neuromolecular Med. 2003;3(2):65-94; Mody I, et al., Trends Pharmacol Sci. 1995; Oct.; 16(10):356-9; Zheng Z. et al., Curr Mol Med. 2003 Jun.;3(4):361-72). Thus, there exists in the art the need for novel methods to locally block calcium flux in the brain, in order to minimize apoptotic neuronal cell death immediately after stroke and traumatic head injury.